Study On Transformation Of Cauliflower (Brassica Oleracea Var.botrytis) With Bt Gene

Cauliflower (Brassica oleracea Var. botrytis) was planted widely in china, its planting area was multiplied rapidly and varieties were increased year after year owning to its abundant nutrition and special flavor. Now, cauliflower was provided in markets all year-around and acted an important role in vegetable production in china. Nevertheless. some lepidopters which damage crucifer severely, such as cabbageworm larva (Pieris rapae Linne). diamondback moth larva (Plutella xylostella Linne) and sugarbeet moth larva (Spodoptera exigua hubner) etc, distributed universally and decreased the yield and quality of cauliflower greatly. Consequently, they restrict anniversary production of cauliflower badly.Plant genetic engineering open a new way for resistant cultivars breeding. Bt genes were derived from Bacillus thuringiensis. its toxin protein could kill lepidopters specifically but was harmless to higher animal and human being. Thereby, it was of potential to obtain insect-resistance resource by introducing Bt gene into cauliflower.In this experiment, we establish a system of regeneration and genetic transformation of cauliflower. Accordingly, Bt gene was transferred into cauliflower by Agrobacterium tumefaciens-medialed transformation and then transgenic plants were obtained. The main results were as follows:1. As for adventitious shoot regeneration of cauliflower, the combination of 6-BA and IAA would get a perfect effect. And hypocotyl was optimal explant compared to cotyledon.2. AgNO3 would inhibit shoot regeneration of cauliflower, while STS would promote shoot regeneration for better absorbance. 29.4μM STS was optimal, and it was harmful in case of excess of it.3.The regeneration frequency of Nan160 was up to 94.45% on the MS medium supplemented with 2.0mg/l 6-BA, and that of Xue60 and Xw55 were up to 81.91% and 77.80% with 1.0mg/1 6-BA, 0.1mg/l IAA and 29.4μM STS. respectively.4. 10mg/l kanamycin would completely inhibit green shoot regeneration fromhypototyl of cauliflower; no shoots except some white shoots were left.5. Cefotaxime put negative effect to shoot regeneration.6. The transformation frequency of cauliflower would be improved by using nursing feeder layer during co-cultivation, and the effect of tobacco cell suspension was better than Acetosyringone's.7. Genomic DNA was extracted from some putative transgenic plants, and the results of PCR and Southern blotting analysis showed that Bt gene was integrated into the plant genome. Some transgenic plants of Nanl60 and Xue60 have been obtained by now.8. The primary result of insect bioassay on eight transformants of Nan 160 and two transformants of Xue60 showed that toxin protein was produced from some of them and the expression levels of Bt gene were different.